Fig. 3.

Analysis of stomatin-complexes after cross-linking with DSS and flotation. Stripped ghosts were cross-linked with (A) 0.8 mM EGS or (B) 0.8 mM DSS. Respective membranes were treated with cold Triton X-100, subjected to flotation, and cross-linked stomatin was identified by Western blotting of density gradient fractions. Note that cross-linked stomatin floats to low density. The dimer (about 65 kDa) is visible as major product. The monomer is not seen due to prolonged electrophoresis (24 h at 70 V). (C) In a different experiment, gradient fractions were analysed for cross-linked stomatin and GLUT1 by Western blotting, as indicated. Note that both cross-linked proteins float to low density. (D) DRM-fractions 3 and 4 from DSS-cross-linked membranes were pooled, solubilised, and stomatin-complexes were isolated by immunoaffinity chromatography. Elution fractions were analysed by SDS-PAGE/silver staining and MS of excised bands, as indicated. (E) Semi-quantitative data of the 500 kDa and 300 kDa complexes are given in AvTIC units. Note the major amount of aquaporin-1 (AQP1) associated with the 300 kDa protein complex. M, marker; 1–5, respective elution fractions.